Therapeutic agents containing cannabis flavonoid derivative for ocular disorders

ABSTRACT

The present invention provides a cannabis-based flavonoid pharmaceutical composition including any one or more selected from the group of Apigenin, Cannflavin A, Cannflavin B, Cannflavin C, Chiysoeriol, Cosmosiin and Flavocannabiside or their synthases, for the prevention and treatment of certain ocular diseases and related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application derives priority from U.S. Provisional PatentApplication No. 62/190,001 filed 8 Jul. 2015.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to flavonoid derivatives and, moreparticularly, to cannabis flavonoid derivatives or the pharmaceuticallyacceptable salt thereof that may be used in a pharmaceutical compositionfor preventing and treating ocular disorders particularly glaucoma andmyopia.

2. Description of the Background

Myopia or Nearsightedness, is the most common refractive error of theeye, and it has become more prevalent in recent years. About 1.6 billionpeople in the world suffered from myopia in 2000 and the number isexpected to rise to 2.5 billion by 2020 (Francisco B M, Salvador M,Amparo N, 2015. Oxidative Stress in Myopia. Oxidative Medicine andCellular Longevity. Vol 2015 ID: 750637). A study by the National EyeInstitute (NEI) shows the prevalence of myopia grew from 25 percent ofthe U.S. population (ages 12 to 54) in 1971-1972 to a whopping 41.6percent in 1999-2004. Though the exact cause for this increase innearsightedness among Americans is unknown, many eye doctors feel it hassomething to do with eye fatigue from computer use and other extendednear vision tasks, coupled with a genetic predisposition for myopia.

Normal and high myopia remains a major medical problem and lead toseveral eye pathologies such as retinal detachment, glaucoma, macularhemorrhage, cataracts and as such causes visual deterioration andeventually blindness. In an attempt to determine the genetic basis ofmyopia, studies have shown that a genetic variant of the BIKE (BMP2K)kinase contributes to high myopia (Liu, H. P., Lin, Y. J., Lin, W. Y.,Wan, L., Sheu, J. J. C., Lin, H. J., & Tsai, F. J. (2009)). A novelgenetic variant of BMP2K contributes to high myopia. Journal of clinicallaboratory analysis, 23(6), 362-367), Li, Zhi-Kui. “Epidemiology,genetics and treatments for myopia. Int Journal of Ophthamology 2011,4(6), This genetic variant has as such been identified as a potentialtherapeutic target for the prevention and correction of myopia.

Flavonoids are common constituents of plants and cover a wide range offunctions including acting as yellow pigments in petals and leaves toattract pollinating insects. They might also appear as bluish pigments(anthocyanins) to receive certain wavelengths of light, which permitsthe plant to be aware of the photoperiod. Many of these flavonoids alsoprotect the plants by being involved in the filtering of harmfulultraviolet light. Some flavonoids play crucial roles in establishingsymbiotic fungi, while at the same time they fight infections caused bypathogenic fungi.

Flavonoids have relevant pharmacological activities such as;antioxidant, antidiabetic, anti-inflammatory, antiallergic, antibiotic,antidiarrheal, CNS and against cancer. In particular administration ofanthocyanoside oligomer appeared to improve subjective symptoms andobjective contrast sensitivity in myopia subjects (Lee, J., Lee, H. K.,Kim, C. Y., Hong, Y. J., Choe, C. M., You, T. W., & Seong, G. J. (2005).Purified high-dose anthocyanoside oligomer administration improvesnocturnal vision and clinical symptoms in myopia subjects. Britishjournal of nutrition, 93(06), 895-899).

Cannabis is credited to have several beneficial pharmacologicalproperties. Unfortunately much attention on Cannabis is focused on itsrecreational use as a psychoactive drug. Studies have identified overtwenty flavonoids in the Cannabis plant, such as; cannflavin A,cannflavin B, cannflavin C, chrysoeril, cosmosiin, flavocannabiside,vitexin, isovitexin, apigenin, kaempferol, myricetin, quercetin,luteolin, homoorientin and orientin (Turner, C. E., Elsohly, M. A., &Boeren, E. G., “Constituents of Cannabis sativa L. XVII., A review ofthe natural constituents”, Journal of Natural Products, 43(2), 169-234(1980). The distribution of these flavonoids in the plant variesdepending on the type of flavonoid. The total content of flavonoids inthe Cannabis' leaves and flowers can reach 1-2.5% of its dry weightdepending on environment factors and the variety of the plant.

Cannabis flavonoids have been shown to have several pharmacologicalproperties. Apart from the specific pharmacologic properties identified,cannabis flavonoids are thought to play synergistic roles with othermetabolites in the plant. For example, some flavonoids are volatile,lipophilic, permeate membranes, and seem to retain pharmacologicalproperties in cannabis smoke (Sauer, M. A., Rifka, S. M., Hawks, R. L.,Cutler, G. B., & Loriaux, D. L., “Journal of Pharmacology andExperimental Therapeutics”, 224(2), 404-407 (1983). Flavonoids maymodulate the pharmacokinetics of THC, via a mechanism shared by CBD, theinhibition of P450 3A11 and P450 3A4 enzymes. These two related enzymesmetabolize environmental toxins from procarcinogens to their activatedforms. P450-suppressing compounds as such serve as chemoprotectiveagents, shielding healthy cells from the activation of benzo[α]pyreneand aflatoxin B1 (Offord, E. A., Mace, K., Avanti, O., & Pfeifer, A. M.,“Mechanisms Involved In The Chemoprotective Effects Of Rosemary ExtractStudied In Human Liver And Bronchial Cells”, Cancer Letters, 114(1),275-281, (1997), which are two procarcinogenic agents found in cannabissmoke (McPartland, J. M., & Pruitt, P. L., “Alternative Therapies InHealth And Medicine”, 5(4), 57 (1999). Cannabis flavonoids thus may bemodulating the therapeutic effects of THC and CBDs by eithersynergistically enhancing desired pharmacologic effects or reducingdetrimental effects.

Given the abundance of evidence supporting the health benefits ofcannabis flavonoids, the present inventors have successfully synthesizedrare cannabis flavonoids including cannflavin A, cannflavin B andcannflavin C, isocannflavin B and their analogs have demonstratedability to inhibit the BMP2K kinase which has implication in thedevelopment of myopia. The present invention relates to the use of thenewly synthesized flavonoids alone or in combination with otherflavonoids or related bioactive compounds particularly the cannabinoidsto treat or prevent ocular diseases shown to be inhibited by the use ofthese flavonoids and the drug combinations.

SUMMARY OF THE INVENTION

It is, therefore, an object of the invention to provide a pharmaceuticalcomposition for the prevention and treatment of ocular diseases usingspecific cannabis-based flavonoid compounds.

It is another object to provide a method for isolating specificcannabis-based flavonoid pharmaceutical compositions from raw plantmaterial that are biologically active in the prevention and treatment ofocular disease.

It is still another object to provide a method for synthesizing saidspecific cannabis-based flavonoid pharmaceutical compositions.

In accordance with the foregoing objects, the present invention providesa flavonoid-based pharmaceutical composition for the prevention andtreatment of ocular disease having the structure of the general formulaof FIG. 1 or a pharmaceutically acceptable salt thereof.

Wherein,

R1-R10 may be any one or more substituents selected from the groupconsisting of a hydrogen molecule (H), a hydroxide molecule (OH), amethyl group comprising one carbon atom bonded to three hydrogen atoms(CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl),Bromine (Br), Fluorine (F), Glutamic acid (Glu), geranyl chain, prenylchain and any salts or derivatives of the foregoing. A and B may each beeither a single or double bond.

A method for the prevention and treatment of ocular disease is alsodisclosed using the specific cannabis-based flavonoid pharmaceuticalcompositions above is also disclosed, as well as a method for isolatingthe specific flavonoid-based pharmaceutical compositions from raw plantmaterial, and a method for synthesizing said flavonoid-basedpharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features, and advantages of the present invention willbecome more apparent from the following detailed description of thepreferred embodiments and certain modifications thereof when takentogether with the accompanying drawings in which:

FIG. 1 is an illustration of the general cannabis-based flavonoidpharmaceutical compositions according to the present invention.

FIG. 2 is the structure of the specific cannabis-based flavonoidpharmaceutical composition.

FIG. 3 is the dose response curve for the inhibition of BIKE (BMP2K)kinase by cannabis flavonoid derivatives

FIG. 4 is the structures of related cannabis flavonoid derivatives.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to preferred embodiment of thepresent invention, examples of which are illustrated in the accompanyingdrawing.

The present invention is a group of cannabis-based flavonoidpharmaceutical compositions selected from among the group of Apigenin,Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin andFlavocannabiside useful for the prevention and treatment of certainocular diseases.

The cannabis-based flavonoid pharmaceutical composition for theprevention and treatment of diseases has the structure of the generalformula of FIG. 1 or a pharmaceutically acceptable salt thereof.

Wherein,

R1-R10 may be any one or more substituents selected from the groupconsisting of a hydrogen molecule (H), a hydroxide molecule (OH), amethyl group comprising one carbon atom bonded to three hydrogen atoms(CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl),Bromine (Br), Fluorine (F), Glutamic acid (Glu), a geranyl chain orprenyl chain and any salts or derivatives of the foregoing. A and B mayeach be either a single or double bond.

The most preferred structure of the synthesized flavonoid presented inFIG. 2.

In an embodiment, a method for the prevention and treatment of oculardisorders using the specific cannabis-based flavonoid pharmaceuticalcompositions above is also disclosed. Administration may be by variousroutes including oral, rectal or intravenous, epidural muscle,subcutaneous, intrauterine, or blood vessels in the brain(intracerebroventricular) injections. The flavonoid derivatives of thegeneral and specific formulas (FIGS. 1-2) according to the presentinvention and a pharmaceutically acceptable salt thereof may beadministered in an effective dose, depending on the patient's conditionand body weight, extent of disease, drug form, route of administration,and duration, within a range of from 0.1 to 500 mg between 1-6 times aday. Of course, most dosages will be by a carrier. The specific doselevel and carrier for patients can be changed according to the patient'sweight, age, gender, health status, diet, time of administration, methodof administration, rate of excretion, and the severity of disease.

The composition may be formulated for external topical application, oraldosage such as powders, granules, tablets, capsules, suspensions,emulsions, syrups, aerosols, suppositories, or in the form of a sterileinjectable solution. Acceptable carriers and excipients may compriselactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate,calcium silicate, cellulose, methyl cellulose, microcrystallinecellulose, polyvinylpyrrolidone, water, methyl benzoate, propylbenzoate, talc, magnesium stearate, and mineral oil.

Bioactivity of the above-described compounds has been verified by use ofkinase inhibition assays to determine the effect of the cannabisflavonoids in the onset and progression of ocular disorders. Theinhibition of BIKE (BMP2K) kinases in particular has been shown to be atherapeutic target that could block the onset and progression of oculardisorders.

Kinase Inhibition Assay

Cannabis flavonoids and their analogs were subjected to kinaseinhibition assay. The compounds were first screened at a singleconcentration of 10 μM in the primary assay. Compounds inhibiting atleast 70% of specific kinases were subjected to further screening todetermine kd values. To determine the kd values, competition bindingassays were established, authenticated and executed as describedpreviously (Fabian et al., 2005, Karaman et al., 2008). For most assays,kinases were fused to T7 phage strains (Fabian et al. 2005) and for theother assays, kinases were produced in HEK-293 cells after which theywere tagged with DNA for quantitative PCR detection (data not shown). Ingeneral, full-length constructs were used for small, single domainkinases, and catalytic domain constructs for large multi-domain kinases.The binding assays utilized streptavidin-coated magnetic beads treatedwith biotinylated small molecule ligands for 30 minutes at roomtemperature which generated affinity resins for the kinase assays. Theliganded beads were blocked with excess biotin and washed with blockingbuffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to removeunbound ligand and to reduce non-specific phage binding. Bindingreactions were assembled by combining kinases, liganded affinity beads,and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05%Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100%DMSO and diluted directly into the assay (Final DMSOconcentration=2.5%). All reactions were performed in polypropylene384-well plates in a final volume of 0.04 ml. The assay plates wereincubated at room temperature with shaking for 1 hour and the affinitybeads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beadswere then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μMnon-biotinylated affinity ligand) and incubated at room temperature withshaking for 30 minutes. The kinase concentration in the eluates wasmeasured by quantitative PCR. Kd values were determined using a standarddose response curve using the hill equation. Curves were fitted using anon-linear least square fit with the Levenberg-Marquardt algorithm.

A method for isolating the specific cannabis-based flavonoidpharmaceutical compositions from raw plant material is also disclosed.The Cannabis flavonoid and derivatives were isolated and synthesized asdescribed in PCT application PCT/US15/62331.

FIG. 3 is the dose response curve for the inhibition of BIKE (BMP2K)kinase by cannabis flavonoid derivatives

FIG. 4 illustrates the structure of closely related cannabis flavonoidderivatives considered to be within the scope and spirit of theinvention.

It should now be apparent that the above-described invention provides apharmaceutical composition for the prevention and treatment of diseasewith specific cannabis-based flavonoid derivatives a method for theprevention and treatment of disease using the specific cannabis-basedflavonoid pharmaceutical compositions.

It is to be understood, therefore, that the invention may be practicedotherwise than as specifically set forth in the appended claims.

1. A flavonoid pharmaceutical composition, having a chemical structureas shown below, or any pharmaceutically acceptable salt thereof:

wherein, R₃, R₅, R₆, R₇, and R₁₀ are none, one, or more substituentsindependently selected from the group consisting of: a hydrogen atom, ahydroxyl group, a methyl group, an alkoxy group, a carboxyl group,chlorine, bromine, fluorine, glutamic acid, a geranyl chain, a prenylchain, and any salt of the foregoing.
 2. The composition of claim 1,further comprising a carrier substance selected from the groupconsisting of: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,erythritol, maltitol, starches, gum acacia, alginate, gelatin, calciumphosphate, calcium silicate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinylpyrrolidone, water, methylbenzoate, propyl benzoate, talc, magnesium stearate, and mineral oil. 3.The composition of claim 1, wherein the composition is formulated in aform selected from the group consisting of: a powder, a granule, atablet, a capsule, a suspension, an emulsion, a syrup, an aerosol, asuppository, or an injectable solution.
 4. A method for treatment of anocular disorder, comprising administering to a patient the compositionaccording to claim
 1. 5. The method for treatment of an ocular disorderaccording to claim 4, wherein said ocular disorder comprises glaucoma.6. The method for treatment of an ocular disorder according to claim 4,wherein said ocular disorder comprises myopia.
 7. A method for treatmentof an ocular disorder, comprising: administering to a patient, acompound having a chemical structure as shown below, or anypharmaceutically acceptable salt thereof:

wherein, R₃, R₅, R₆, R₇, and R₁₀ are none, one, or more substituentsindependently selected from the group consisting of: a hydrogen atom, ahydroxyl group, a methyl group, an alkoxy group, a carboxyl group,chlorine, bromine, fluorine, glutamic acid, a geranyl chain, a prenylchain, and any salt of the foregoing.
 8. The method of claim 7, whereinthe ocular disorder comprises glaucoma or myopia.
 9. The method of claim7, wherein the administration to the patient of the compound comprisesadministering the compound via a route selected from the groupconsisting of: a topical route, an oral route, and a rectal route. 10.The method of claim 7, wherein the administration to the patient of thecompound comprises injecting the compound into a location of thepatient's body selected from the group consisting of: a vein, anepidural muscle, a subcutaneous location, an intrauterine location, andan intracerebroventricular location.
 11. The method of claim 7, furthercomprising: selecting a dose of the compound based on at least onefactor selected from the group consisting of: the patient's condition,the patient's body weight, an extent of the ocular disorder, a form ofthe compound, a route of administration of the compound, and a durationof administration; wherein the administration to the patient of thecompound comprises administering the selected dose of the compound. 12.The method of claim 7, further comprising: selecting a dose of thecompound; wherein the administration to the patient of the compoundcomprises administering between 1 to 6 of the selected doses of thecompound each day.
 13. The method of claim 7, further comprising:selecting a dose of the compound within a range from 0.1 milligrams (mg)to 500 mg; wherein the administration to the patient of the compoundcomprises administering the selected dose of the compound.
 14. Themethod of claim 7, wherein the patient has not been diagnosed with theocular disorder, and wherein the administration to the patient of thecompound is a preventative treatment.